Effect of monoclonal antibodies against von Willebrand factor and platelet
glycoproteins IIb/IIIa on the platelet retention test
J McPherson, S Brownlea and MB Zucker
The platelet retention test provides a measure of the number of platelets
retained in a column of glass beads and is one of the few in vitro platelet
function tests that is abnormal in von Willebrand's disease (vWd). In a
two-stage test, 1 mL of blood (designated A) was passed through the column,
followed by 5 mL of isotonic saline and then 5 mL of blood (B) in which
platelet retention was measured. With normal blood as A and B, retention is
very high in all 5 mL of blood B. In the first stage, platelets adhere to
the glass beads; this requires fibrinogen but not von Willebrand factor
(vWf). The platelet-platelet adhesion in the second stage requires vWf, is
dependent on release of ADP, and fails to occur if thrombasthenic platelets
are tested. Retention was normal when blood from a patient with
afibrinogenemia was used as blood B. We have now used monoclonal antibodies
to elucidate further the mechanism of platelet retention. Five antibodies
to different epitopes on vWf essentially abolished retention in the one-
stage test and in the second stage of the two-stage test, but had no effect
on the first stage. Thus, the entire vWf molecule must be free of antibody
to function in the platelet-platelet adhesion of the second stage of this
test. Binding of the antigen-antibody complex to the platelet Fc receptor
was not responsible, as Fab and F(ab')2 fragments of one of the antibodies
were as effective as intact antibody, and as neither heat-aggregated IgG
nor a polyclonal antibody to plasma factor IX inhibited retention. F(ab')2
fragments of 6D1, an antibody to platelet GP Ib that prevents binding of
vWf to platelets, also inhibited the second phase of retention. An antibody
that inhibits binding of fibrinogen and vWf to GP IIb/IIIa (LJ-CP8)
inhibited both the first and second stages of retention, whereas LJ-P5, an
antibody that inhibits only the binding of vWf to GP IIb/IIIa, caused
slight inhibition of retention when normal or afibrinogenemic blood was
used as blood B and was reported to cause only partial inhibition of ADP-
induced platelet aggregation in this afibrinogenemic patient. The results
suggest that vWf is altered during rapid passage of blood through the
glass-bead column so that it attaches to GP Ib, exposing GP IIb/IIIa, which
then binds the altered vWf or fibrinogen, either of which can induce
platelet aggregation (platelet-platelet adhesion) and thus retention in the
column.
Volume 70,
Issue 2,
pp. 546-550,
08/01/1987
Copyright © 1987 by The American Society of Hematology
